CN111518752B

CN111518752B - Application of vitamin D3 and analogues thereof in promoting differentiation of human skin fibroblasts into adipocytes

Info

Publication number: CN111518752B
Application number: CN202010266244.XA
Authority: CN
Inventors: 张传茂; 王向阳
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2020-04-07
Filing date: 2020-04-07
Publication date: 2021-12-10
Anticipated expiration: 2040-04-07
Also published as: CN111518752A

Abstract

The invention provides an application of vitamin D3 and an analogue thereof in promoting human skin fibroblasts to differentiate into adipocytes, which is to pre-treat human skin fibroblasts with vitamin D3 or an analogue thereof with a final concentration of 10-100nM before the human skin fibroblasts are subjected to in vitro induced differentiation. On the basis of the original 6+2+8 induced differentiation mode, the invention obviously improves the efficiency of differentiating the human skin fibroblasts into the adipocytes by the pretreatment of the vitamin D3 or the analogues thereof. The method can efficiently induce and differentiate human skin fibroblasts into adipocytes, and provides a new thought and a new method for clinical skin wound healing, scar repair and treatment of lipodystrophy.

Description

Application of vitamin D3 and analogues thereof in promoting differentiation of human skin fibroblasts into adipocytes

Technical Field

The invention relates to the technical field of biology, in particular to application of vitamin D3 and analogues thereof in promoting human skin fibroblasts to differentiate into adipocytes.

Background

CN201910876049.6 discloses a method for inducing human skin fibroblasts to differentiate into adipocytes in vitro, which has relatively low efficiency of differentiating human skin fibroblasts into adipocytes, and is difficult to be widely used in clinical applications such as wound repair, scar repair, and lipodystrophy treatment.

Disclosure of Invention

The invention aims to provide application of vitamin D3 and analogues thereof in promoting differentiation of human skin fibroblasts into adipocytes.

Another object of the present invention is to provide a method for efficiently inducing the differentiation of human skin fibroblasts into adipocytes in vitro.

To achieve the object of the present invention, in a first aspect, the present invention provides the use of vitamin D3 and its analogs in promoting the differentiation of human skin fibroblasts into adipocytes.

The application comprises the following steps: human skin fibroblasts are pre-treated with vitamin D3 or an analogue thereof at a final concentration of 10-100nM (preferably 10nM or 50nM) before being subjected to in vitro induced differentiation.

In the present invention, the analogue of vitamin D3 may be Calcipotriol (Calcipotriol).

In a second aspect, the present invention provides a method for inducing human skin fibroblasts to differentiate into adipocytes in vitro, comprising culturing human skin fibroblasts in vitro in a basal medium until the confluency of the cells reaches 50-70%, adding vitamin D3 or its analogue (calcipotriol) at a final concentration of 10-100nM (preferably 10nM or 50nM), pretreating the cells for 12-48 hours, and then inducing differentiation;

the step of inducing differentiation comprises: changing the culture medium into an induced differentiation culture medium I, culturing for 6-10 days, and changing the culture medium every two days; then, replacing the induced differentiation culture medium I with an induced differentiation culture medium II, and culturing for 2-4 days; finally, changing the induced differentiation culture medium to a basic culture medium, culturing for 8-14 days, and changing the culture medium once every two days;

wherein the basic culture medium is a high-glucose DMEM culture medium containing 10-20% fetal calf serum, 100U/mL penicillin and 100 mu g/mL streptomycin;

the induced differentiation culture medium I is a basic culture medium containing 0.5-1 mu M dexamethasone, 10-20 mu g/mL insulin, 1-2 mu M rosiglitazone and 0.2-0.5mM 1-methyl-3-isobutyl xanthine;

the induction differentiation culture medium (II) is a basal culture medium containing 10-20 mug/mL of insulin.

Preferably, the basal medium is a high-glucose DMEM medium containing 20% fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin.

Preferably, the differentiation induction medium (i) is a basal medium containing 1. mu.M dexamethasone, 20. mu.g/mL insulin, 2. mu.M rosiglitazone and 0.5mM 1-methyl-3-isobutylxanthine.

Preferably, the differentiation induction medium (c) is a basal medium containing 20. mu.g/mL of insulin.

The culture medium is preferably prepared at present, so as to avoid degradation of effective components of the medicine.

In one embodiment of the present invention, a method for inducing human skin fibroblasts to differentiate into adipocytes in vitro comprises: culturing human skin fibroblasts in vitro in a basal medium until the number of the cultured cells reaches 50-70%, transferring the cultured cells to less than 10 generations (preferably 2-7 generations, and more preferably 7 generations), adding vitamin D3 or its analogues with a final concentration of 10-100nM (preferably 10nM or 50nM), and pretreating the cells for 24 hours; then, the culture medium is changed into an induced differentiation culture medium I, and the culture is carried out for 6 days, and the liquid is changed every two days; then, the induced differentiation medium I is replaced by an induced differentiation medium II, and the culture is carried out for 2 days; finally, changing the induced differentiation culture medium to a basic culture medium, culturing for 8 days, and changing the culture medium once every two days;

wherein the basic culture medium is a high-glucose DMEM culture medium containing 20% fetal calf serum, 100U/mL penicillin and 100 mu g/mL streptomycin;

the induced differentiation culture medium I is a basic culture medium containing 1 mu M dexamethasone, 20 mu g/mL insulin, 2 mu M rosiglitazone and 0.5mM 1-methyl-3-isobutyl xanthine;

the induction differentiation culture medium is a basal culture medium containing 20 mu g/mL insulin.

In the present invention, the cell culture conditions are: 35-37 ℃ and 5% CO₂Preferably 37 ℃ and 5% CO₂。

In a third aspect, the present invention provides adipocytes prepared in accordance with the above-described methods.

In a fourth aspect, the invention provides any one of the following uses of the adipocytes:

(1) used for preparing biomedical materials and tissue repair materials;

(2) for wound repair and scar repair;

(3) can be used for treating lipodystrophy.

The cell source used in the invention is primary human skin fibroblast (HDF), and the material taking and the separation are convenient. The cell isolation method is as follows: first, skin tissue having a diameter of 1mm was removed from the skin of volunteers and washed 3 times with Phosphate Buffered Saline (PBS); then, excess adipose tissues were removed with an ophthalmic scissors in a clean bench, and the remaining tissues were minced and washed 3 times with PBS; then, 1-2mL of pancreatin 0.25% was added to immerse all tissue pieces in a solution containing 5% CO₂Digesting for 5-10min at 37 ℃ in the cell culture box; after digestion was complete, 1mL of basal medium was added to stop digestion. Then, the pipette gun is used to blow and suck the cell clusters gently and repeatedly. Finally, the cell suspensions were transferred to 35mm petri dishes, 4mL of basal medium was added, and the mixture was placed in a cell incubator under 5% CO₂Culturing at 37 deg.C for 4-6 days. During the culture process, the culture medium can be supplemented appropriately according to the amount of the culture medium in the culture dish. Finally, the isolated cells were primary human dermal fibroblasts. After the cells are full, subculture is carried out. In the whole cell separation process, aseptic operation is ensured, and pollution is avoided. After the cells are separated, mycoplasma detection is carried out, and the cells can be used for subsequent experiments after the cells are determined to be free from mycoplasma pollution. We have performed multiple induction differentiation experimentsThe generation number of HDF cells for inducing differentiation is not too high, and is preferably within 10 generations; HDF cells over 10 generations induce differentiation less efficiently, and the higher the generation number, the harder the induction of differentiation, and the lower the differentiation efficiency. Therefore, HDF cells having a low number of generations should be cryopreserved as much as possible in order to facilitate the subsequent induced differentiation experiment.

And (3) inducing a differentiation scheme: the whole process of inducing and differentiating HDF cells mainly comprises two parts of pretreatment and induced differentiation. First, vitamin D3(1, 25-dihydrovitamin D3, Calcitriol) or its analog Calcipotriol (Calcipotriol) was pretreated for 24 hours when the degree of confluence of HDF cells reached 50-70%. Then, induced differentiation is carried out according to a differentiation mode of '6 +2+ 8-16 days', namely, a first induced differentiation medium is induced for 6 days, a second induced differentiation medium is induced for 2 days, and a basic medium is continuously cultured for 8 days. The mode of pretreating with vitamin D3 or its analogues and inducing differentiation greatly improves the efficiency of human skin fibroblasts to differentiate into adipocytes.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

on the basis of the original 6+2+8 induced differentiation mode, the invention obviously improves the efficiency of differentiating human skin fibroblasts into adipocytes by the pretreatment of vitamin D3 or an analogue thereof (calcipotriol). Taking calcipotriol as an example, firstly, human fibroblasts are pretreated with 10nM calcipotriol for 24 hours, then induced differentiation is carried out according to a mode of '6 +2+ 8', the number of finally obtained fat cells is 2.5 times that of a control group (not pretreated with calcipotriol), and the expression quantity of fat cell marker genes (Adiponectin, Fabp4 and Leptin) is improved by 2 times. The method can efficiently induce and differentiate human skin fibroblasts into adipocytes, and provides a new thought and a new method for clinical skin wound healing, scar repair and treatment of lipodystrophy.

Drawings

FIG. 1 shows the results of oil red staining induced by differentiation of human dermal fibroblasts into adipocytes in example 1 of the present invention.

FIG. 2 is the statistical result of the oil red staining induced by the differentiation of human dermal fibroblasts into adipocytes in example 1 of this invention.

FIG. 3 shows the results of detecting marker genes of adipocytes obtained by induced differentiation in example 1 of the present invention.

FIG. 4 shows the results of oil red staining induced by differentiation of human dermal fibroblasts into adipocytes in example 2 of this invention.

FIG. 5 shows the statistical results of the oil red staining induced by differentiation of human dermal fibroblasts into adipocytes in example 2 of the present invention.

FIG. 6 shows the results of detecting marker genes of adipocytes obtained by induced differentiation in example 2 of the present invention.

FIG. 7 shows the results of oil red staining induced by differentiation of human dermal fibroblasts into adipocytes in example 3 of this invention.

FIG. 8 shows the statistical results of the oil red staining induced by the differentiation of human dermal fibroblasts into adipocytes in example 3 of this invention.

FIG. 9 shows the results of detecting marker genes of adipocytes obtained by induced differentiation in example 3 of the present invention.

FIG. 10 shows the results of oil red staining induced by differentiation of human dermal fibroblasts into adipocytes in example 4 of this invention.

FIG. 11 shows the statistical results of the oil red staining induced by the differentiation of human dermal fibroblasts into adipocytes in example 4 of this invention.

FIG. 12 shows the results of examining marker genes of adipocytes obtained by induced differentiation in example 4 of the present invention.

In fig. 2-3, 5-6, 8-9, 11-12, P <0.001, P <0.0001, the differences between the different treatment groups were statistically significant.

Detailed Description

The invention provides a method for efficiently inducing human skin fibroblasts to differentiate into adipocytes in vitro. The method comprises the following steps:

1. taking out the cover glass soaked in 75% alcohol, burning and sterilizing on an alcohol lamp, and then paving in 35mm culture dishes, wherein 4 cover glass are paved on each culture dish;

2. adding 4ml of basic culture medium into the culture dish, and spreading the overlapped cover glass with forceps for later cell passage;

3. after the primary HDF cells in the 10cm culture dish were overgrown, the culture medium in the culture dish was aspirated off, washed 2 times with 2ml of Phosphate Buffered Saline (PBS) each time; then sucking off PBS, adding 1ml of pancreatin with the concentration of 0.25%, and repeatedly shaking the culture dish by two hands to enable the pancreatin to uniformly infiltrate the bottom of the culture dish; finally placing in a container containing 5% CO₂Digesting for 1-2min at 37 ℃ in the cell culture box;

4. the digested cells were removed and 0.5ml basal medium was added to the culture dish to stop the digestion; then repeatedly blowing and sucking by using a pipettor, uniformly blowing and transferring the cells into a 1.5ml centrifuge tube, and centrifuging for 4 minutes at 1000 rpm/min;

5. sucking off the supernatant, adding 1ml of a basal medium, slightly and repeatedly sucking, and uniformly mixing the cells; then, according to the following steps of 1: 4, evenly dividing the cell suspension into 4 35mm culture dishes paved with cover glass, repeatedly shaking the culture dishes by two hands to uniformly mix the cells, and then putting the cells in a cell culture box for culture;

6. after the confluence of the cells reaches 50-70%, adding vitamin D3 with the final concentration of 10-100nM or calcipotriol analogue of 10-100nM, and pretreating the cells for 24 hours;

7. sucking off the basic culture medium, adding 3ml of induced differentiation culture medium I, putting the culture medium back into the cell culture box for continuous induced culture for 6 days, and replacing the fresh culture medium every two days; when the culture medium is replaced, the culture medium is gently added along the wall of the culture dish;

8. absorbing the induced differentiation culture medium I, adding 3ml of induced differentiation culture medium II, and carrying out induced culture for 2 days; as the induced differentiation proceeded, a shiny circular fat drop was found in the cells under a 10-fold microscope;

9. absorbing the induced differentiation culture medium II, adding 3ml of basal culture medium, continuing to culture for 8 days, and replacing the fresh culture medium every two days; under a microscope, cells containing fat drops are more and more, and fat drops in the cells are larger and larger;

10. after the induction differentiation is finished, taking out the cover glass in the culture dish for oil red staining, and detecting the induction differentiation effect; in addition, RNA of the residual cells of the culture dish is extracted, fluorescent quantitative PCR is carried out, the expression of marker genes Adiponectin, Fabp4 and Leptin of the mature adipocytes is detected, and the induced differentiation result is further verified.

Detection of induced differentiation results:

oil red dyeing

Oil red O is a very strong fat solvent and dye agent, capable of binding triglycerides and is therefore frequently used for fat dyeing. We performed oil red staining on cells after induced differentiation to detect fat droplets within mature adipocytes.

The specific operation is as follows:

1) the coverslip was washed 3 times with PBS and then fixed with 4% Paraformaldehyde (PFA) for 20 minutes;

2) after 3 washes with PBS, equilibrate with 60% isopropanol for 5 minutes;

3) hermetically dyeing with oil red O (dissolved in 60% isopropanol) at a concentration of 3g/L for 10 minutes;

4) washing with 60% isopropanol for 3 times;

5) staining with hematoxylin for 2 minutes;

6) washing with PBS for 3 times, air drying, and sealing to form image;

7) and counting the number of cells which are positively stained by oil red.

Detection of mature adipocyte marker gene

Adipoectin, Fabp4, and Leptin are marker genes for mature adipocytes, and are often used to measure the effect of adipocyte differentiation induction. Trizol is used for extracting total RNA from cells after induced differentiation, and after reverse transcription, fluorescent quantitative PCR is carried out to detect the expression levels of three genes, namely Adiponectin, Fabp4 and Leptin.

The PCR primer sequences used were as follows:

Adiponectin:5’-GATGGCAGAGATGGCAC-3’

5’-GCTGAGCGGTATACATAGG-3’

Fabp4:5’-ACGAGAGGATGATAAACTGGTGG-3’

5’-GCGAACTTCAGTCCAGGTCAAC-3’

Leptin:5’-CACCAAAACCCTCATCAAGACA-3’

5’-CTTTCTGTTTGGAGGAGACTGACT-3’

the following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

The media used in the examples below:

the basal medium was high-glucose DMEM medium containing 20% fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin.

The induced differentiation culture medium (I) is a basic culture medium containing 1 mu M dexamethasone, 20 mu g/mL insulin, 2 mu M rosiglitazone and 0.5mM 1-methyl-3-isobutyl xanthine.

High-glucose DMEM medium was purchased from Gibco.

Example 1 method for inducing differentiation of human skin fibroblasts into adipocytes with high efficiency in vitro

This example illustrates the results of induced differentiation using a 10nM vitamin D3(Calcitriol) pretreatment for 24 hours.

The induced differentiation method is as follows:

1. the isolated primary HDF cells were passaged to a coverslipped 35mm petri dish, 3ml basal medium was added, and the dish was placed in a medium containing 5% CO₂Culturing at 37 ℃ in the cell culture box;

2. after the confluence degree of the cells reaches 50-70%, adding vitamin D3 with the final concentration of 10nM, and pretreating the cells for 24 hours;

3. sucking off the basic culture medium, adding an induced differentiation culture medium I, continuously carrying out induced culture for 6 days, and replacing a fresh culture medium every two days;

4. absorbing the induced differentiation culture medium I, adding the induced differentiation culture medium II, and carrying out induced culture for 2 days;

5. absorbing the induced differentiation culture medium, adding a basic culture medium, continuously culturing for 8 days, and replacing the fresh culture medium every two days.

After induction differentiation is finished, the result of induction differentiation is detected through oil red staining and mature fat cell marker gene detection. The results are as follows:

oil red dyeing result

As shown in fig. 1, the red part is stained fat, and the non-induced HDF cells have no fat accumulation, while the HDF cells induced to differentiate for 16 days have significant fat accumulation. Compared with a control group (without vitamin D3 pretreatment), the induction of vitamin D3 with 10nM for 24 hours can obviously promote the differentiation of fat cells.

② statistical result of oil red dyeing

As shown in FIG. 2, the number of cells showing positive staining for oil red in the non-induced HDF cells was 0, and the number of cells showing positive staining for oil red in the HDF cells induced to differentiate for 16 days were: 130 pieces/cm in the control group²10nM vitamin D3 pretreatment group 271/cm²。

③ detection of mature adipocyte marker gene

As shown in FIG. 3, by quantitative PCR (quantitative PCR), marker genes of three mature adipocytes, i.e., Adiponectin, Fabp4, and Leptin, were detected. The expression of three marker genes of the 10nM vitamin D3 pretreatment group was significantly higher than that of the control group.

Example 2 method for inducing differentiation of human skin fibroblasts into adipocytes with high efficiency in vitro

This example illustrates the results of induced differentiation using 50nM vitamin D3(Calcitriol) pretreatment for 24 hours.

The method for inducing differentiation was the same as in example 1, but the "6 +2+ 8" mode for inducing differentiation was also used. Except that the pretreatment was changed to 50nM vitamin D3 for 24 hours.

oil red dyeing result

As shown in fig. 4, the red part is stained fat, and the non-induced HDF cells have no fat accumulation, while the HDF cells induced to differentiate for 16 days have significant fat accumulation. Compared with a control group, the induction of vitamin D3 with the concentration of 50nM for 24 hours can obviously promote the differentiation of the fat cells.

② statistical result of oil red dyeing

As shown in FIG. 5, the number of cells showing positive staining for magenta in the non-induced HDF cells was 0, and the number of cells showing positive staining for magenta in the HDF cells induced to differentiate for 16 days were: control group (without vitamin D3 pretreatment) 118/cm²50nM vitamin D3 pretreatment group 542/cm²。

③ detection of mature adipocyte marker gene

As shown in FIG. 6, by quantitative PCR (quantitative PCR), marker genes of three mature adipocytes, i.e., Adiponectin, Fabp4, and Leptin, were detected. The expression of three marker genes of the 50nM vitamin D3 pretreated group is significantly higher than that of the control group.

Example 3 method for inducing human skin fibroblasts to differentiate into adipocytes with high efficiency in vitro

This example illustrates the results of induced differentiation using 10nM Calcipotriol (Calcipotriol) as an example of 24 h pretreatment.

The method for inducing differentiation was the same as in example 1, but the "6 +2+ 8" mode for inducing differentiation was also used. Except that the pretreatment was changed to 10nM calcipotriol for 24 hours.

oil red dyeing result

As shown in fig. 7, the red portion is stained fat, and the non-induced HDF cells did not accumulate fat, whereas the HDF cells induced to differentiate for 16 days had significant fat accumulation. Compared with a control group (without calcipotriol pretreatment), the induction of the 10nM calcipotriol after 24 hours of pretreatment can obviously promote the differentiation of the fat cells.

② statistical result of oil red dyeing

As shown in FIG. 8, oil Red in uninduced HDF cellsThe number of cells staining positive was 0, and the number of cells staining positive for oil red in HDF cells induced to differentiate for 16 days was: control group 115 pieces/cm²10nM calcipotriol pretreatment group 250/cm²。

③ detection of mature adipocyte marker gene

As shown in FIG. 9, by quantitative PCR (quantitative PCR), marker genes of three mature adipocytes, i.e., Adiponectin, Fabp4, and Leptin, were detected. The expression of three marker genes of the 10nM calcipotriol pretreatment group is obviously higher than that of the control group.

Example 4 method for inducing human skin fibroblasts to differentiate into adipocytes with high efficiency in vitro

This example illustrates the results of induced differentiation using 50nM Calcipotriol (Calcipotriol) pretreatment for 24 hours.

The method for inducing differentiation was the same as in example 1, but the "6 +2+ 8" mode for inducing differentiation was also used. Except that pretreatment was changed to 50nM calcipotriol for 24 hours.

oil red dyeing result

As shown in fig. 10, the red portion is stained fat, and the non-induced HDF cells have no fat accumulation, while the HDF cells induced to differentiate for 16 days have significant fat accumulation. Compared with a control group (without calcipotriol pretreatment), 50nM calcipotriol can obviously promote adipocyte differentiation after 24 hours of pretreatment.

② statistical result of oil red dyeing

As shown in FIG. 11, the number of cells showing positive staining for magenta in the non-induced HDF cells was 0, and the number of cells showing positive staining for magenta in the HDF cells induced to differentiate for 16 days were: control group 122/cm²523/cm in 50nM Calcipotriol pretreatment group²。

③ detection of mature adipocyte marker gene

As shown in FIG. 12, by quantitative PCR (quantitative PCR), marker genes of three mature adipocytes, i.e., Adiponectin, Fabp4, and Leptin, were detected. The expression of three marker genes of the 50nM calcipotriol pretreatment group is obviously higher than that of the control group.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A method for inducing human skin fibroblasts to differentiate into adipocytes in vitro is characterized in that the human skin fibroblasts are cultured in vitro in a basal culture medium, vitamin D3 or calcipotriol with the final concentration of 10-100nM is added when the confluence degree of the cells reaches 50-70%, the cells are pretreated for 12-48 hours, and then induced differentiation is carried out;

2. The method according to claim 1, wherein the differentiation induction medium (r) is a basal medium containing 1 μ M dexamethasone, 20 μ g/mL insulin, 2 μ M rosiglitazone and 0.5mM 1-methyl-3-isobutylxanthine.

3. The method of claim 1, wherein the differentiation-inducing medium (c) is a basal medium containing 20 μ g/mL of insulin.

4. The method of claim 1, wherein the final concentration of vitamin D3 or calcipotriol is 10nM or 50 nM.

5. The method of claim 1, wherein human skin fibroblasts are cultured in vitro in a basal medium and passed through within 10 passages, and after the confluence of the cells reaches 50-70%, vitamin D3 or calcipotriol is added to the cells at a final concentration of 10-100nM, and the cells are pretreated for 24 hours; then, the culture medium is changed into an induced differentiation culture medium I, and the culture is carried out for 6 days, and the liquid is changed every two days; then, the induced differentiation medium I is replaced by an induced differentiation medium II, and the culture is carried out for 2 days; finally, changing the induced differentiation culture medium to a basic culture medium, culturing for 8 days, and changing the culture medium once every two days;

6. The method of any one of claims 1 to 5, wherein the cell culture conditions are: 35-37 ℃ and 5% CO₂。

7. The method of claim 6, wherein the cell culture conditions are: 37 ℃ and 5% CO₂。